Angiotensin II (Ang II) exerts important physiologic and pathophysiologic effects on cardiovascular and renal systems through its AT1 receptor subtype. Emerging evidence indicates that AT2 receptor subtype also mediates significant biological responses such as natriuresis, anti-growth, anti-proliferation, and pro-apoptosis. These inhibitory actions appear to antagonize the well-known stimulatory actions of AT1 receptor. Because a delicate balance between these opposing actions must be maintained for health and because these actions are all governed by the corresponding stimulatory and inhibitory signals, elucidation of inhibitory signaling mechanisms becomes more and more important in biomedicine and may hold keys for innovative therapeutics.Unlike the AT1 receptor for which the stimulatory signaling mechanisms are well studied, signaling and activation (R*) mechanisms of the inhibitory AT2 receptor remain unknown. Our results indicate that AT2 receptors directly activate SH2 domain containing phosphatase- 1 (SHP- 1) and b1y2 proteins, respectively, with novel mechanisms. AT2 may produce constitutively active state (R*), Ang Il-induced R*, and DTT-regulated R*. These R* states triggers activation of multiple signal effector pathways. Therefore, we hypothesize that AT2 is a novel inhibitory receptor capable of activating inhibitory signals with distinct mechanisms through multiple R* states.
The specific Aims are:1. Determine the molecular basis for physical association between SHP-1 and the AT2 receptor.2. Determine the molecular basis for physical association between gb1y2 subunits and the AT2 receptor.3. Understand activation mechanisms of the inhibitory AT2 receptor.Many techniques such as mutagenesis, co-immunoprecipitation, Western blotting, peptide binding, mammalian two-hybrid system, in vitro translation, luminescence, and BRET will be used in this project. Various cell lines (CHO-K1, L cells, and NI E- 115) and primary rabbit proximal tubular epithelial cells will be used accordingly for this project. Stable cell lines will be generated. Gq/ill chimera protein and jellyfish photoprotein apoaequorin will be used extensively as reporter system to detect AT2 receptor activation. In this proposal, tools of bioinformatics and molecular modeling will also be used to assist experimental design and interpretation. We believe this proposed study will provide important insight into mechanisms of AT2-triggered inhibitory signals and AT2 activation. This proposal has the potential to open up novel avenues for therapy of hypertrophy and fibrosis.
